The present invention is directed to compounds which are useful as inhibitors of metalloproteases, e.g. zinc proteases, particularly zinc hydrolases, and which are effective in the prophylaxis and treatment of disease states which are associated with vasoconstriction of increasing occurrences. Examples of such disorders are high blood pressure, coronary disorders, cardiac insufficiency, renal and myocardial ischaemia, renal insufficiency, dialysis, cerebral ischaemia, cardiac infarct, migraine, subarachnoid hemorrhage, Raynaud syndrome and pulmonary high pressure. In addition the compounds are useful as cytostatic and cerebroprotective agents for inhibition of graft rejection, for organ protection and for treatment of ophthalmological diseases.
Endothelins are peptides, that exist in three isoforms ET-1, ET-2, and ET-3, each encoded by a distinct gene. They have been originally discovered in the conditioned medium of porcine endothelial cells in 1988 by Yanagisawa (Yanagisawa M; Kurihara H; Kimura S; Tomobe Y; Kobayashi M; Mitsui Y; Yazaki Y; Goto K; Masaki T: A novel potent vasoconstrictor peptide produced by vascular endothelial cells [see comments]. NATURE (Mar. 31, 1988), 332(6163), 411-5.). The active ETs are peptides of 21 amino acids with two intramolecular disulfide bridges. They are produced from preproproteins of 203 to 212 amino acids which are processed by furin like endopeptidases to the biologically inactive big-endothelin (big-ET). The big-ETs are specifically processed to mature ETs by a hydrolytic cleavage between amino acids 21 and 22 that are Trp21-Val22 (big-ET-1, big ET-2) and Trp21-Ile22 in big-ET-3 respectively. Already in 1988 a specific metalloprotease was postulated to be responsible for this specific cleavage. In 1994 ECE-1 (endothelin converting enzyme-1) was purified and cloned from bovine adrenal (Xu D, Emoto N, Giaid A, Slaughter C, Kaw S, de Witt D, Yanagisawa M: ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell (1994) 78: 473-485.).
ECE-1 is a membrane bound type II zinc-endopeptidase with a neutral pH optimum and a zinc binding motif HExxHx( greater than 20)E. It belongs to subfamily M13 and has a large 681 amino acid ectodomain that comprises the active site. Other members of the M13 family are NEP24.11 (neutral endopeptidase), PEX, a phosphate regulating neutral endopeptidase, and Kell blood group protein that has recently been described as a big-ET-3 processing enzyme. Members of the M13 family of human origin are characterized by a high molecular weight ( greater than 80 kDa) a number of conserved disulfide bridges and a complex glycosylation pattern. The structure of NEP has recently been solved. (Oefner et al, J. Mol. Biol. 2000, 296, 341-349). The catalytic domain of ECE and related human M13 proteinases are significantly larger ( greater than 650 amino acids) than members of matrix metalloproteases (MMPs). Unlike the family of the MMPs which belong to the metzincins and display a typical HExxHxxGxxH pattern members of the M13 family are gluzincins comprising a HExxHx( greater than 20)E pattern. These two families are clearly different in size of catalytic domains, structure and zinc coordinating pattern of ligands. Active sites of the two families show clear differences which has clear impact on type of inhibitors and the potential selectivity.
Therefore one aspect of the present invention is to provide compounds useful for the selective inhibition of ECE- 1.
The present invention relates to a compound of formula (I): 
wherein
R1 is hydrogen, alkylcarbonyl, or arylcarbonyl;
R2 is alkyl, alkylcycloalkyl, alkylcycloalkylalkyl, cycloalkyl, halogenalkyl, carboxyalkyl, aminoalkyl, dialkylaminoalkyl, alkoxyalkyl, alkoxycarbonylalkyl, alkinyl, aryl, arylalkyl, arylalkyl(alkoxycarbonyl) alkyl, arylcarbonylalkyl, aryloxyalkyl, arylalkenyl, aryl( alkoxycarbonyl)alkyl, heteroaryl, heteroarylalkyl, heterocyclyl or hetercycylalkyl;
R3 is hydrogen, aryl, alkyl, or arylalkyl, arylsulfonyl, heteroarylsulfonyl;
R4 is hydrogen, arylalkyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, heteroarylsulfonyl, carboxyalkyl, carboxyalkylsulfonyl, or alkoxycarbonylalkyl; or the groups xe2x80x94NR3R4 or R5xe2x80x94[Nxe2x80x94N(R4)]xe2x80x94R3 form a saturated or unsaturated 5- or 6-membered aliphatic ring;
R5 is hydrogen, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, heteroarylalkylcarbonyl, heterocyclyl, (mono- or di-alkylamino)-alkylcarbonyl, (mono- and dialkyl)aminosulfonyl, arylaminocarbonyl, alkyl alkylcarbonyl, alkoxycarbonyl, aryl, arylalkyl, arylalkoxycarbonyl, or heteroaryl;
R6 is hydrogen, alkyl, aryl, or carboxyalkyl;
X is xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94NHxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)NR6 or C(O)xe2x80x94Oxe2x80x94, or a pharmaceutically acceptable esteror pharmaceutically acceptable salt thereof.
The term xe2x80x9calkylxe2x80x9d as used herein, alone or in combination, means a straight-chain or branched-chain alkyl group containing a maximum of 7, preferably a maximum of 4, carbon atoms, e.g., methyl, ethyl, n-propyl, 2-methylpropyl (iso-butyl), 1-methylethyl (iso-propyl), n-butyl, and 1,1-dimethylethyl (t-butyl).
The term xe2x80x9ccarboxyxe2x80x9d refers to the group xe2x80x94C(O)OH.
The term xe2x80x9ccarbamoylxe2x80x9d refers to the group xe2x80x94C(O)NH2.
The term xe2x80x9ccarbonylxe2x80x9d refers to the group xe2x80x94C(O)xe2x80x94.
The term xe2x80x9chalogenxe2x80x9d refers to the group xe2x80x9cfluoro, bromo, chloro and iodo, preferably fluoro and/or chloro, most preferably fluoro.
The term xe2x80x9csulfonylxe2x80x9d refers to the group xe2x80x94S(O2)xe2x80x94.
The term xe2x80x9calkenylxe2x80x9d refers to a hydrocarbon chain as defined for alkyl having at least one olefinic double bond (including for example, vinyl, allyl and butenyl).
The term xe2x80x9calkinylxe2x80x9d refers to a hydrocarbon chain as defined for alkyl having at least one olefinic triple bond (including for example propinyl, butin-(1)-yl, etc.
The term xe2x80x9calkoxyxe2x80x9d, alone or in combination, means an alkyl ether group in which the term xe2x80x98alkylxe2x80x99 has the significance given earlier, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec.butoxy, tert.butoxy and the like.
The term xe2x80x9calkoxycarbonylxe2x80x9d refers to refers to a group of the formula xe2x80x94C(O)Rc wherein Rc is alkoxy as defined above.
The term xe2x80x9chydroxyxe2x80x9d refers to the group xe2x80x94OH, the term xe2x80x9ccyanoxe2x80x9d to the group xe2x80x94CN.
The term xe2x80x9chydroxyalkylxe2x80x9d means an alkyl group as defined earlier which is substituted by a hydroxy group.
The term xe2x80x9cthioalkylxe2x80x9d refers to an alkyl group as defined above which is substituted by a xe2x80x94SH group.
The term xe2x80x9chalogenalkylxe2x80x9d refers to an alkyl group as defined earlier which is substituted by one to three halogen atoms, preferably fluoro, e.g. trifluoromethyl, 2,2,2-trifluoroethyl, etc.
xe2x80x9cCarboxyalkylxe2x80x9d means a lower-alkyl as defined above which is substituted by a HOOC-group.
The term xe2x80x9calkylcarbonylxe2x80x9d, alone or in combination, means an acyl group derived from an alkanecarboxylic acid, i.e. alkyl-C(O)xe2x80x94, such as acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl etc.
The term xe2x80x9ccycloalkylxe2x80x9d, alone or in combination, signifies a saturated, cyclic hydrocarbon group with 3-8, preferably 3-6 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and the like.
The term xe2x80x9caminoxe2x80x9d refers to the group xe2x80x94NH2.
The term xe2x80x9carylxe2x80x9d for R2xe2x80x94 alone or in combination-, refers to an aromatic carbocyclic radical, i.e. a 6 or 10 membered aromatic or partially aromatic ring, e.g. phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl or naphthyl, and most preferably phenyl. The aryl moiety is optionally substituted with one or more groups, preferably 1-5, more preferably 1-3, independently selected from halogen, preferably fluor, alkoxycarbonyl, e.g. methylcarbonyl, carboxy, cyano, alkyl, alkoxy, phenyl, phenoxy, trifluormethyl, trifluormethoxy, more preferably fluor, alkoxycarbonyl, alkyl, trifluoromethyl and trifluoromethoxy and most preferably fluor. The most preferred aromatic groups are naphthyl or phenyl substituted with one or more fluor atoms, e.g. naphthyl, 2,3,4,5,6-pentafluorophenyl or biphenyl.
The term xe2x80x9carylxe2x80x9d for R3 and R6xe2x80x94 alone or in combinationxe2x80x94refers to an aromatic carbocyclic radical, i.e. a 6 or 10 membered aromatic or partially aromatic ring, e.g. phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl or naphthyl, and most preferably phenyl. The aryl moiety is optionally substituted with one or more groups, preferably 1-5, more preferably 1-3, independently selected from halogen, preferably fluor, alkoxycarbonyl, e.g. methylcarbonyl, carboxy, cyano, alkyl, alkoxy, phenyl, phenoxy, trifluormethyl, trifluormethoxy, hydroxy, alkylamido, e.g. acetamido, nitro, alkylsulfonyl, e.g. methylsulfonyl, more preferably alkyl or alkoxy.
The term xe2x80x9carylxe2x80x9d for R4xe2x80x94 alone or in combinationxe2x80x94, refers to an aromatic carbocyclic radical, i.e. a 6 or 10 membered aromatic or partially aromatic ring, e.g. phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl or naphthyl, and most preferably phenyl. The aryl moiety is optionally substituted with one or more groups, preferably 1 to 3, independently selected from halogen, preferably fluor, alkoxycarbonyl, e.g. methylcarbonyl, carboxy, cyano, alkyl, alkoxy, phenyl, phenoxy, trifluormethyl, trifluormethoxy, cyclohexyl, hydroxy, alkylamido, e.g. acetamido, nitro, alkylsulfonyl, e.g. methylsulfonyl, more preferably fluor, chlor, brom, alkoxy, carboxy, alkoxycarbonyl, and most preferably fluor. Examples for aromatic groups are phenyl, 2,4,5-trifluorophenyl, and 2,4-difluorophenyl.
The term xe2x80x9carylxe2x80x9d for R5xe2x80x94 alone or in combinationxe2x80x94, refers to an aromatic carbocyclic radical, i.e. a 6 or 10 membered aromatic or partially aromatic ring, e.g. phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl or naphthyl, and most preferably or phenyl. The aryl moiety is optionally substituted with one or more groups, preferably 1-5, more preferably 1-3, independently selected from halogen, preferably fluor, alkoxycarbonyl, e.g. methylcarbonyl, carboxy, cyano, alkyl, alkoxy, phenyl, phenoxy, trifluormethyl, trifluormethoxy, more preferably alkyl or alkoxy, e.g. methyl or methoxy. Examples for these aryl groups are 4-methyl-phenyl and 4-methoxy-phenyl.
The term xe2x80x9caryloxyxe2x80x9d refers to an aryl group as defined above attached to a parent structure via an oxy radical, i.e., aryl-Oxe2x80x94.
The term xe2x80x9cheteroarylxe2x80x9d for R4xe2x80x94 alone or in combinationxe2x80x94refers to an aromatic monovalent mono- or bicyclic radical having 5 to 10, preferably 5 to 6 ring atoms, containing one to three heteroatoms, preferably one heteroatom, e.g. independently selected from nitrogen, oxygen or sulfur. Examples of heteroaryl groups are thiophenyl, isoxazolyl, thiazolyl, pyridinyl, pyrrolyl, imidazolyl, tetrazolyl, preferably pyridinyl, isoxazolyl and thiazolyl. Optionally, the heteroaryl group can be mono-, di- or tri-substituted, independently, with phenyl, alkyl, alkylcarbonyl, alkoxycarbonyl, hydroxy, amino, alkylamino, dialkylamino, carboxy, alkoxycarbonylalkyl, preferably alkyl.
The term xe2x80x9cheteroarylxe2x80x9d for R3 or R5xe2x80x94 alone or in combinationxe2x80x94refers to an aromatic monovalent mono- or bicyclic radical having 5 to 10, preferably 5 to 6 ring atoms, containing one to three heteroatoms, preferably one heteroatom, e.g. independently selected from nitrogen, oxygen or sulfur. Examples of heteroaryl groups are pyridinyl, thiophenyl, isoxyzolyl, isoquinolyl, quinolyl, indolyl, pyrimidine, pyridazine, and pyrazine, preferably thiophenyl, furanyl, pyrrolidinyl, indolyl and isoxazolyl. Optionally, the heteroaryl group can be mono-, di- or tri-substituted, independently, with phenyl, alkyl, alkylcarbonyl, alkoxycarbonyl, hydroxy, amino, alkylamino, dialkylamino, carboxy, oxo, alkoxycarbonylalkyl, preferably alkyl.
The term xe2x80x9cheterocyclylxe2x80x9dxe2x80x94alone or in combinationxe2x80x94refers to a non-aromatic monovalent mono- or bicyclic radical having 5 to 10, preferably 5 to 6 ring atoms, containing one to three heteroatoms, preferably one heteroatom, e.g. independently selected from nitrogen, oxygen or sulfur. Optionally the heterocyclic ring can be substituted by a group independently selected from halogen, alkyl, alkoxy, oxocarboxy, alkoxycarbonyl, etc. and/or on a secondary nitrogen atom (i.e. xe2x80x94NHxe2x80x94) by alkyl, arylalkoxycarbonyl, alkylcarbonyl or on a tertiary nitrogen atom (i.e. xe2x95x90Nxe2x80x94) by oxido. Examples for heterocyclic groups are morpholinyl, pyrrolidinyl, piperidyl, etc.
The term xe2x80x9cdimeric formxe2x80x9d means a compound wherein the two R1 groups of two identical compounds of formula I have been replaced by a common single bond or wherein R1 is glutathione-Sxe2x80x94 or cysteine-Sxe2x80x94 or ester and/or alkylcarbonyl or arylcarbonyl derivatives thereof, e.g. acetylcysteine-Sxe2x80x94 or benzoylcysteine-Sxe2x80x94, preferably glutathione-Sxe2x80x94, cysteine-Sxe2x80x94, acetylcysteine-Sxe2x80x94 or benzoylcysteine-Sxe2x80x94.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. The salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxylic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. In addition these salts may be prepared form addition of an inorganic base or an organic base to the free acid. Salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium salts and the like. Salts derived from organic bases include, but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polymine resins and the like.
xe2x80x9cPharmaceutically acceptable estersxe2x80x9d means that compounds of general formula (I) may be derivatised at functional groups to provide derivatives which are capable of conversion back to the parent compounds in vivo. Examples of such compounds include physiologically acceptable and metabolically labile ester derivatives, such as methoxymethyl esters, methylthiomethyl esters and pivaloyloxymethyl esters. Additionally, any physiologically acceptable equivalents of the compounds of general formula (I), similar to the metabolically labile esters, which are capable of producing the parent compounds of general formula (I) in vivo, are within the scope of this invention.
The compounds of formula (I) and their salts and esters are useful in inhibiting mammalian metalloprotease activity, particularly zinc hydrolase activity. More specifically, the compounds of formula (I) and their salts and esters are useful as medicaments for the treatment and prophylaxis of disorders which are associated with diseases caused by endothelin-converting enzyme (ECE) activity. Inhibiting of this enzyme would be useful for treating myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma. In addition the compounds are useful as cytostatic and cerebroprotective agents, for inhibition of graft rejection, for organ protection and for treatment of ophthalmological diseases.
In more detail, the present invention relates to a compound of formula (I) 
wherein
R1 is hydrogen, alkylcarbonyl, or arylcarbonyl;
R2 is alkyl, alkylcycloalkyl, alkylcycloalkylalkyl, cycloalkyl, halogenalkyl, carboxyalkyl, aminoalkyl, dialkylaminoalkyl, alkoxyalkyl, alkoxycarbonylalkyl, alkinyl, aryl, arylalkyl, arylalkyl(alkoxycarbonyl)alkyl, arylcarbonylalkyl, aryloxyalkyl, arylalkenyl, aryl(alkoxycarbonyl)alkyl, heteroaryl, heteroarylalkyl, heterocyclyl or hetercycylalkyl;
R3 is hydrogen, aryl, alkyl, or arylalkyl, arylsulfonyl, heteroarylsulfonyl;
R4 is hydrogen, arylalkyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, heteroarylsulfonyl, carboxyalkyl, carboxyalkylsulfonyl, or alkoxycarbonylalkyl; or the groups xe2x80x94NR3R4 or R5xe2x80x94[Nxe2x80x94N(R4)]xe2x80x94R3 form a saturated or unsaturated 5- or 6-membered aliphatic ring;
R5 is hydrogen, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, heteroarylalkylcarbonyl, heterocyclyl, (mono- or di-alkylamino)-alkylcarbonyl, (mono- and dialkyl)aminosulfonyl, arylaminocarbonyl, alkyl, alkylcarbonyl, alkoxycarbonyl, aryl, arylalkyl, arylalkoxycarbonyl, or heteroaryl;
R6 is hydrogen, alkyl, aryl, or carboxyalkyl;
X is xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94NHxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)NR6 or C(O)xe2x80x94Oxe2x80x94 or a dimeric form, or a pharmaceutically acceptable ester, or a pharmaceutically acceptable salt thereof, preferably a pharmaceutically acceptable ester, or a pharmaceutically acceptable salt thereof, and most preferably a pharmaceutically acceptable salt thereof.
In a preferred embodiment, the invention refers to compounds wherein R1 is hydrogen.
In a further preferred embodiment of the present invention R2 is alkyl, halogenalkyl, alkylamino, alkoxy, cycloalkyl, cycloalkylamino, aryl, arylalkyl, aryloxy, arylalkylamino, arylalkoxy, heteroaryl, amino, or (mono- and dialkyl)amino; more preferably alkyl, halogenalkyl, alkylamino, alkoxy, cycloalkyl, cycloalkylamino, aryl, arylalkyl, or heteroaryl and even more preferably is aryl or heteroaryl and most preferably aryl. The term aryl in the definition for R2 especially means naphthyl or phenyl, wherein phenyl is optionally substituted by one or more fluor or by one phenyl group, e.g. R2 is naphthyl, 2,3,4,5,6-pentafluorophenyl or biphenyl.
According to the present invention R3 is preferably hydrogen or alkyl, most preferably hydrogen.
In the above compounds R4 is preferably hydrogen, arylalkyl, alkyl, arylsulfonyl, heteroarylsulfonyl, cycloalkylalkyl, or carboxyalkyl, more preferably hydrogen, alkyl, arylalkyl, cycloalkyl, arylsulfonyl, or carboxyalkyl, most preferably hydrogen, alkyl, cycloalkyl, carboxyalkyl or arylalkyl, even more preferably hydrogen, alkyl or arylalkyl, e.g. hydrogen, 2,4,5-trifluorobenzyl, 2,4-difluorobenzyl, benzyl, methyl, ethyl, isopropyl, isobutyl, benzyl or HO2Cxe2x80x94CH2xe2x80x94, or cycloalkylpropylmethyl.
In a preferred embodiment of the present invention R5 is hydrogen, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, aryl, arylalkyl, arylcarbonyl, (mono- and dialkylamino)alkylcarbonyl, (mono- and dialkyl)aminosulfonyl, arylalkoxycarbonyl, arylaminocarbonyl, arylsulfonyl, heteroarylcarbonyl, heteroarylalkylcarbonyl, heteroarylsulfonyl, arylaminocarbonyl, heteroaryl, or heterocyclyl, more preferably aryl, arylalkyl, arylcarbonyl, arylalkoxy, arylaminocarbonyl, arylsulfonyl, heteroarylcarbonyl, heteroarylalkylcarbonyl, heteroarylsulfonyl, arylaminocarbonyl, heteroaryl, or heterocyclyl, more preferably arylsulfonyl, arylalkyl, heteroarylalkylcarbonyl, heteroarylsulfonyl and most preferably 4-methyl-benzenesulfonyl, benzyl, 4-methoxybenzenesulfonyl, (1H-indol-3-yl)acetyl, thiophene-2-yl, or 3,5-dimethyl-isoxyzolyl-4-sulfonyl.
In the above described compounds X is preferably xe2x80x94SO2xe2x80x94, xe2x80x94C(O)xe2x80x94, and most preferably xe2x80x94SO2xe2x80x94.
In the most preferred embodiment of the present invention, the compounds maybe described by the formula (II) 
wherein R1, R2, R3, R4, R5 and X are as defined above and pharmaceutically acceptable esters and/or salts thereof.
In a further preferred embodiment of the present invention R1 is hydrogen, R2 is naphthyl or phenyl, wherein phenyl is optionally substituted by one or more fluor or by one phenyl group, R3 is hydrogen or alkyl, R4 is hydrogen, alkyl or arylalkyl, R5 is arylsulfonyl, arylalkyl, heteroarylalkylcarbonyl, heteroarylsulfonyl; and X is xe2x80x94SO2xe2x80x94.
Preferred embodiments of the present invention are the compounds exemplified in the examples. Especially the present invention comprises compounds according to formula (I) or (II)
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-isobutyl-Nxe2x80x2-(4-methyl-benzenesulfonyl)-hydrazide;
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-(4-methyl-benzenesulfonyl)-hydrazide;
(2S,4R)/4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-benzyl-hydrazide;
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-benzyl-Nxe2x80x2-(4- methyl-phenylsulfonyl)-hydrazide;
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-methyl-Nxe2x80x2-(4-methyl-benzenesulfonyl)-hydrazide;
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-benzenesulfonyl-hydrazide;
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-(4-methoxy-benzenesulfonyl)-hydrazide;
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-[(1H-indol-3-yl)-acetyl]-hydrazide;
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-thiophene-2-sulfonyl-hydrazide;
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-(3,5-dimethyl isoxazole-4-sulfonyl)-hydrazide;
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-cyclopropylmethyl-Nxe2x80x2-(4-methyl-benzenesulfonyl)-hydrazide;
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-(4-methyl-benzenesulfonyl)-Nxe2x80x2-(2,4,5-trifluoro-benzyl)-hydrazide;
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-(2,5-difluoro-benzyl)-Nxe2x80x2-(4-methyl-benzenesulfonyl)-hydrazide
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-isopropyl-Nxe2x80x2-(4-methyl-benzensulfonyl)-hydrazide;
(2S,4R)-[Nxe2x80x2-[4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carbonyl]-N-(4-methyl-benzenesulfonyl)-hydrazino]-acetic acid;
(2S,4R)-1-(Biphenyl-4-sulfonyl)-4-mercapto-pyrrolidine-2-carboxylic acid Nxe2x80x2-methyl-Nxe2x80x2-(4-methyl-benzenesulfonyl)-hydrazide;
(2S,4R)-4-Mercapto-1-(2,3,4,5,6-pentafluoro-benzenesulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-(4-methyl-benzenesulfonyl)-hydrazide;
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid Nxe2x80x2-benzyl-Nxe2x80x2-(4-methoxy-benzenesulfonyl)-hydrazide;
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid N-methyl-Nxe2x80x2-(4-methyl-benzenesulfonyl)-hydrazide; and
(2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid N-methyl-Nxe2x80x2-benzyl-Nxe2x80x2-(4-methyl-benzenesulfonyl)-hydrazide.
These compounds show IC50 values in the radioimmunoassay (E on ECE-inhibition, see below) of about 50 nM to 1 xcexcM.
The invention also refers to pharmaceutical compositions containing a compound as defined above and a pharmaceutically acceptable excipient.
A further embodiment of the present invention refers to the use of compounds as defined above as active ingredients in the manufacture of medicaments comprising a compound as defined above for the prophylaxis and treatment of disorders which are caused by endothelin-converting enzyme (ECE) activity especially myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection.
Further the invention refers to the use of compounds as described above for the treatment or prophylaxis of diseases which are associated with myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection.
In addition the invention comprises compounds as described above for use as therapeutic active substances, in particular in context with diseases which are associated with zinc hydrolase activity such as myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection.
The invention also comprises a method for the therapeutic and/or prophylactic treatment of myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection, which method comprises administering a compound as defined above to a human being or animal.
The invention also relates to the use of compounds as defined above for the inhibition of zinc hydrolase activity.
The invention relates also to a process for the preparation of a compound as defined above comprising reaction of a compound of formula III 
wherein
R1, R2, and X are as defined above and A is a HS-protecting group with HNR3NR4R5 for introduction of a hydrazide: or
HNR3NR4R5 with R5 as protecting group followed by conversion or introduction of R3 and R4; optionally followed by conversion of a R5 and/or R2xe2x80x94X group into a different R5 and/or R2xe2x80x94X group and/or deprotection and or thiol liberation and wherein R3, R4 and R5 are as defined above.
The invention also refers to the above compounds whenever manufactured by a process as described.
The compounds of formula (I) can be prepared by methods known in the art or as described below.
Unless otherwise indicated, the substituents R1, R2, R3 R4, R5, R6, and X are as described above. In the schemes below, all starting materials are known or can be prepared by known methods. 
Step a) of scheme 1 describes the persilylation of hydroxy- and amino groups, e.g. by reaction of compound 1 with hexamethyldisilazan/140xc2x0 C. followed by reaction with R2SO2Cl in THF or conversion to all other R2X described later or di-t-butyldicarbonate/NaHCO3 in dioxane/H2O (BOC protection). For inversion of the configuration (via mesylate) the resulting alcohol 2 is treated with MeSO3H/Ph3P/DIAD in toluene (room temperature to 80xc2x0 C.) or (via bromide) with LiBr/DEAD/Ph3P in THF (4xc2x0 C. to room temperature) or (via chloride) with Ph3P/CCl4 in CH2Cl2 (3xc2x0 C. to room temperature). In case of retention of the configuration (via mesylate) alcohol 2 can be transformed to a compound of formula 3 by reaction with MeSO2Cl/pyridine/DMAP (0xc2x0 C. to room temperature).
For the introduction of a protected thiol moiety, compounds of formula 3 are treated with e.g. triphenylmethanethiol or 4-methoxybenzylmercaptane and K-Ot-Bu in DMF (for Br: 0xc2x0 C. to room temperature; for Cl: 0xc2x0 C.; for Mesylate: room temperature to 100xc2x0 C.). Hydrolysis of ester 4 with aqueous LiOH in THF (0xc2x0 C. to RT) gives acid 5.
The synthesis of final compounds are shown in scheme 2:
The synthesis starts with a preactivation af acid 1a (N-hydroxy-2-pyridone, N,N-dicyclohexylcarbodiimide, 4-Ethylmorpholine in CH2Cl2 at RT) followed by reaction with an alkyl-hydrazine (NHR3NHR4) (step a) or for carboxylic acid hydrazide 2 (R3, R4, R5xe2x95x90H), ester 1b is directly treated with hydrazine (NH2NH2.H2O) in EtOH (at RT). Conversion to the free thiol 3 is done in the following way: in case PG (protecting group) is Tr by reaction with e.g. TFA/Et3SiH at 0xc2x0 C. to room temperature or, in case PG is PMB, by reaction with e.g. TFA/Et3SiH, at 0 to 80xc2x0 C. (step b).
In the case of carboxylic acid hydrazide 2 (R3, R4, R5xe2x95x90H), R4 is introduced by reductive amination: Imine formation with an aldehyde in EtOH followed by reduction with NaBH3CN in THF gives compound 4 (step c). For the introduction of a new R5 in case R4 is an alkyl (R5xe2x95x90H), reaction with ClCOR5, ClCO2R5, ClSO2R5 or ClSO2NR5, iPr2NEt or Huenig""s base CH2Cl2 in the precence of a catalytic amount of DMAP or DMAP-poly or R5NCO in THF at room temperature gives compound 4 which is deprotected to the final thiol 5 as described above (step c and b).
Selective BOC deprotection of compound 2 or 4 (TFA, CH2Cl2 at 0xc2x0 C.), followed by reaction with ClCO2R2, NEM or iPr2NEt, CH2Cl2 or R2NCO in THF at 0xc2x0 C. to room temperature (or conversion to all other R2X described for R5xe2x80x94 introduction above) gives compound 7 (step e). Thiol deprotection as described above gives the final thiol 8 (step b). 
Scheme 3 shows a different way for the synthesis of hydrazides.
Preactivation af acid 1 (N-hydroxy-2-pyridone, N,N-dicyclohexylcarbodiimide, 4-Ethylmorpholine in CH2Cl2 at RT) followed by reaction with an alkyl-hydrazinecarboxylic acid benzyl ester (NHR3NHR4) (step a) gives hydrazide 2 which is converted with HBr in AcOH at 0xc2x0 C. to 3 (step b). A direct conversion from 1 to 3 with preactivation and reaction with NHR3NR4 R5 is possible too (step c). Introduction of a new R3 is done with an R3-halogenide/ NaH in DMF (at 0xc2x0 C. to RT; - greater than 4, step e). Deprotection to the thiol 5 is done in the following way: in case PG is Trby reaction with e.g. TFA/Et3SiH at 0xc2x0 C. to room temperature or, in case PG is PMB, by reaction with e.g. TFA/Et3SiH, at 0 to 80xc2x0 C. (step d). 
Scheme 4 shows an other way for the synthesis of hydrazides.
Preactivation af acid 1 (N-hydroxy-2-pyridone, N,N-dicyclohexylcarbodiimide, 4-Ethylmorpholine in CH2Cl2 at RT) followed by reaction with a tert-butyl 2-alkyl-hydrazinecarboxylate (NHR3NHBOC) (step a) gives hydrazide 2 which by treatment with triethylsilane in TFA at 0 to 80xc2x0 C. gives thiol 3 (step b). Alkylation with alkylhalogenide (R5-halogenide) and DMF with NaH as base (at 0xc2x0 C. to RT) results in compound 4 which gives after Et3SiH/TFA deprotection (as described in scheme 1) thiol 5 (step c, d).
Selective BOC-deprotection (TFA in CH2Cl2- greater than 6) followed by reaction with ClCO2R4, ClSO2R4, iPr2NEt or NEM in CH2Cl2 in the precence of a catalytic amount of DMAP or DMAP-poly at room temperature gives compounds 7 (R4xe2x95x90R5) and 8 which are separated and deprotected (Et3SiH/TFA as described in scheme 1) to thiol 5 (step e, f and d). The not fully substituted hydrazide 8 can be further alkylated (R5-halogenide and DMF/NaH, 0xc2x0 C. to RT) and deprotected (Et3SiH/TFA as described in scheme 1) to the thiol 5 (step g and d). 
Scheme 5 shows further transformation of hydrazide 1. Acylation with xcex3-Bromo-alkanoyl chloride in the presence of iPr2EtN in THF (0xc2x0 C. to RT) gives compound 2 which is cyclised (NaH in DMF at RT). Separation of the two isomers and deprotection of the thiol (Et3SiH/TFA as described in scheme 1) gives hydrazides 3 and 4. 
For the preparation of compounds of formula 5 the reaction pathway of scheme 6 can be followed: the synthesis of the starting material 1 from hydroxyproline is described in scheme 1. TFA/triisopropyl deprotection at reflux for 30 minutes gives thiol 2 that is attached to the resin. The final R2X is introduced either at the beginning or after manipulations at NR3NR4R5 (scheme 9). In the second case, R2X (xe2x95x90BOC) of starting acid 1 is transformed by methods known in the art and described for example in xe2x80x9cThe Practice of Peptide Synthesisxe2x80x9d, M. Bodanszky and A. Bodanszky, Springer Verlag, Berlin, 1984 to a nonacid labile protecting group (e.g. R2Xxe2x95x90FMOC, step a: first selective BOC-deprotection with 40% TFA in CH2Cl2 at RT followed by reaction with Fmoc-OSu in dioxane/water and NaHCO3 as base). 
The resin is prepared as follows (step b): The linker 4-(xcex1,xcex1-diphenylhydroxymethyl)benzoic acid is activated using TPTU, DIEA in DMF and added to benzhydrylamine resin 3. The resin is then treated with thiol 2 in CH2Cl2/TFA to give the resin loaded starting material 5. 
The synthesis of final compounds on resin 1 is shown in scheme 7: The synthesis starts with a preactivation af acid 1 (TPTU, Huenig""s base in DMF at RT) followed by reaction with an alkyl-hydrazine (NH2NHR4) (step a) to give intermediates 2a, 2b or 4 (R3, R4, R5xe2x95x90H, step c). Detachment of the resin to the free thiol 3 is done with TFA/iPr3SiH in CH2Cl2 at RT (step b). In the case of carboxylic acid hydrazide 4, the introduction of a new R5 is done by reaction with ClCOR5, ClCO2R5, ClSO2R5 or ClSO2NR5, in DMF to give compound 2 which is optionally alkylated (alkyl halgenide/DBU in DMF) to the disubstituted hydrazide 6 (step d and f). Detachment of the resin as described above gives the final thiol 3. In the case of reaction of hydrazide 4 with ClSO2R5, double sulfonylation to compound 2a and 2b takes place (step d), these compounds can be separated after detachment from the resin as the corresponding thiol 3.
If R2X is FMOC: Deprotection of compound 2 (20% piperidine/DMF then reaction with ClCO2R2, pyridine, DMF or R2NCO in DMF at RT or conversion to all other R2X described for R4xe2x80x94 introduction above) gives compound 5 (step e). Alkylation (alkyl halgenide/DBU in DMF) and resin deprotection as described above gives the final thiol 3 (step f and b).
The ability of the compounds of formula (I) to inhibit metalloprotease activity, particularly zinc hydrolase activity, may be demonstrated by a variety of in vitro and in vivo assays known to those of ordinary skill in the art.
A) Cell Culture
A stable human umbilical vein endothelial cell line (ECV304) was cultured in xe2x80x9ccell factoriesxe2x80x9d as described until confluency (Schweizer et al. 1997, Biochem. J. 328: 871-878). At confluency cells were detached with a trypsin/EDTA solution and collected by low speed centrifugation. The cell pellet was washed once with phosphate buffered saline pH 7.0 and stored at xe2x88x9280xc2x0 C. until use.
B) Solubilization of ECE from ECV304 cells
All procedures were performed at 0-4xc2x0 C. if not stated otherwise. The cell pellet of 1xc3x97109 cells was suspended in 50 ml of buffer A (20 mM Tris/HCl, pH 7.5 containing 5 mM MgCl2, 100 xcexcM PMSF, 20 xcexcM E64, 20 xcexcM leupeptin) and sonicated. The resulting cell homogenate was centrifuged at 100,000 gav for 60 minutes. The supernatant was discarded and the resulting membrane pellet was homogenized in 50 ml buffer A and centrifugated as described. The washing of the membrane fraction in buffer A was repeated twice. The final membrane preparation was homogenized in 50 ml of buffer B (buffer A+0.5% Tween 20 (v/v), 0.5% CHAPS (w/v), 0.5% Digitonin (w/v)) and stirred at 4xc2x0 C. for 2 hours. Thereafter the remaining membrane fragments were sedimented as described. The resulting clear supernatant containing the solubilized ECE was stored in 1.0 ml aliquots at xe2x88x92120xc2x0 C. until use.
C) ECE Assay
The assay measured the production of ET-1 from human big ET-1. To measure high numbers of samples an assay performed in 96 well plates was invented. The enzyme reaction and the radioimmunological detection of the produced ET-1 was performed in the same well, using a specifically developed and optimized coating technique.
D) Coating of Plates
Fluoronunc Maxisorp White (code 437796) 96 well plates were irradiated with 1 joule for 30 minutes in a UV Stratalinker 2400 (Stratagene). The 96 well plates were then fill with 300 xcexcl protein A solution (2 xcexcg/ml in 0.1 M Na2CO3 pH 9.5) per well and incubated for 48 hours at 4xc2x0 C. Coated plates can be stored for up to 3 weeks at 4xc2x0 C. until use.
Before use the protein A solution is discarded and the plates are blocked for 2 hours at 4xc2x0 C. with 0.5% BSA in 0.1M Na2CO3, pH 9.5.
Plates were washed with bidestilled water and were ready to perform the ECE assay.
E) Screening Assay
Test compounds are solved and diluted in DMSO. 10 xcexcl of DMSO was placed in the wells, followed by 125 xcexcl of assay buffer (50 mM Tris/HCl, pH 7.0, 1 xcexcM Thiorphan, 0,1% NaN3, 0.1% BSA) containing 200 ng big ET-1. The enzyme reaction was started by the addition of 50 xcexcl of solubilized ECE (diluted in assay buffer 1:30 to 1:60 fold (v/v)). The enzyme reaction was carried out for 30 minutes at 37xc2x0 C. The enzyme reaction was stopped by addition of 10 xcexcl 150 mM ETDA, pH 7.0.
Radioimmunoassay
The ET-1 RIA was performed principally as described earlier (Lxc3x6ffler, B.-M. and Maire, J.-P. 1994, Endothelium 1: 273-286). To plates containing the EDTA stopped enzyme reaction mixture 25 xcexcl of assay buffer containing 20000 cpm (3-(125I)Tyr)-endothelin-1 and 25 xcexcl of the ET specific antiserum AS-3 (dilution in assay buffer 1:1000) was added. Plates were incubated under mixing at 4xc2x0 C. over night. Thereafter, the liquid phase was sucked with a plate washer and plates were washed once with bidestilled water. To the washed plates 200 xcexcl scintillation cocktail (Microscint 40 LSC-Cocktail, Packard, code 6013641) was added and plates were counted for 2 minutes per well in a Topcount.
Standard curves were prepared in plates with synthetic ET-1 with final concentrations of 0 to 3000 pg ET-1 per well. In all plates controls for maximal ECE activity (in the presence of 10 xcexcl DMSO) and for background production of ET-1 immunoreactivity (in the presence of 10 mM EDTA or 100 xcexcM phosphoramidon) were performed. Assays were run in triplicate.
F) Kinetic Assay
The described assay format could be used to determine the kinetic characteristics of the used ECE preparation as well as different ECE inhibitors (i.e. Km, Ki) by variation of the substrate concentration used in the assay.
G) Cell based ECE Assay
Human ECE-1c was stable expressed in MDCK cells as described (Schweizer et al. 1997, Biochem. J. 328: 871-878). Cells were cultured in 24 well plates to confluency in Dulbecco""s modified Eagles""s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 0.8 mg/ml geneticin, 100 i.u./ml penicillin and 100 xcexcg/ml streptomycin in a humidified air/CO2 (19:1) atmosphere. Before ECE assay the medium was replaced by 0.5 ml DMEM-HBSS 1:1, 10 mM HEPES pH 7.0 supplemented with 0.1% (w/v) BSA. The inhibitors were added in DMSO at a final concentration of 1%. The enzyme reaction was started by the addition of 0.42 xcexcM human big ET-1 and performed for 1.5 hours at 37xc2x0 C. in an incubator. At the end of incubation, the incubation medium was quickly removed and aliquots were analysed by radioimmunoassay for produced ET-1 as described above.
The ECE screening assay was validated by the measurement of the characteristic inhibitor constants of phosphoramidon (IC50 0.8xc2x10.2 xcexcM) and CGS 314447 (IC50 20xc2x14 nM) [De Lombaert, Stephane; Stamford, Lisa B.; Blanchard, Louis; Tan, Jenny; Hoyer, Denton; Diefenbacher, Clive G.; Wei, Dongchu; Wallace, Eli M.; Moskal, Michael A.; et al. Potent non-peptidic dual inhibitors of endothelin-converting enzyme and neutral endopeptidase 24.11. Bioorg. Med. Chem. Lett. (1997), 7(8), 1059-1064]. The two inhibitors were measured with IC50 values not significantly different from those described in the literature but measured with different assay protocols. In the cell based assay phosphoramidon showed an IC50 of 4 xcexcM. This assay gave additional information about the inhibitory potency of inhibitors under much more physiologic conditions, as e.g. the ECE was embedded in a normal plasma membrane environment. It is important to state, that the screening assay was performed in the presence of 1 xcexcM Thiorphan to block any potential big ET-1 degradation due to the action of NEP24.11. No NEP activity was present in MDCK-ECE-1c transfected cells in preliminary experiments when ET-1 production was measured in presence or absence of thiorphan. In subsequent experiments no thiorphan was added in the incubation medium.
According to the above methods, the compounds of the present invention show IC50 values in the radioimmunoassay (E on ECE-inhibition) of about 50 nM to about 1000 xcexcM. The preferred compounds show values of 50 nM to 1 xcexcM.
As mentioned earlier, medicaments containing a compound of formula I are also an object of the present invention as is a process for the manufacture of such medicaments, which process comprises bringing one or more compounds of formula I and, if desired, one or more other therapeutically valuable substances into a galenical administration form.
The pharmaceutical compositions may be administered orally, for example in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions. Administration can also be carried out rectally, for example using suppositories; locally or percutaneously, for example using ointments, creams, gels or solutions; or parenterally, for example using injectable solutions.
For the preparation of tablets, coated tablets, dragees or hard gelatin capsules the compounds of the present invention may be admixed with pharmaceutically inert, inorganic or organic excipients. Examples of suitable excipients for tablets, dragees or hard gelatin capsules include lactose, maize starch or derivatives thereof, talc or stearic acid or salts thereof.
Suitable excipients for use with soft gelatin capsules include for example vegetable oils, waxes, fats, semi-solid or liquid polyols etc.; according to the nature of the active ingredients it may however be the case that no excipient is needed at all for soft gelatin capsules.
For the preparation of solutions and syrups, excipients which may be used include for example water, polyols, saccharose, invert sugar and glucose.
For injectable solutions, excipients which may be used include for example water, alcohols, polyols, glycerin, and vegetable oils.
For suppositories, and local or percutaneous application, excipients which may be used include for example natural or hardened oils, waxes, fats and semi-solid or liquid polyols.
The pharmaceutical compositions may also contain preserving agents antioxidants, solubilising agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for the variation of osmotic pressure, buffers, coating agents or antioxidants. They may also contain other therapeutically valuable agents.
The dosages in which the compounds of formula I are administered in effective amounts depend on the nature of the specific active ingredient, the age and the requirements of the patient and the mode of application. In general, dosages of 0.1-100 mg/kg body weight per day come into consideration, although the upper limit quoted can be exceeded when this is shown to be indicated.
The following specific examples are provided as a guide to assist in the practice of the invention, and are not intended as a limitation on the scope of the invention.